Cell cultures are used routinely for a variety of research, drug screening and diagnostic testing procedures. Animal and human cell cultures, especially three dimensional cell cultures are used for testing active agents, drugs and cosmetics, as well as for substitution of animal tests and for transplantations.
Neuronal cultures are widely employed in research, particularly in the study of receptor function, receptor localization, neuronal inflammation, neuronal degeneration and cell death. Further, the use of primary neuronal cultures has increased in part due to the increased prevalence of neuronal disorders and diseases such as Huntingtons, Parkinsons and Alzheimers disease in the population. The use of such primary neuronal cultures permit normal and neuropathological target cell populations to be studied in isolation.
Primary cell cultures more faithfully reproduce the characteristics of native tissue than do immortalized cell lines. A serious limitation of using cell lines is that the phenotype and genotype of cells may change with time in culture, especially over several generations. These changes may manifest as a loss of tissue-specific function or biotransformation capacity. Further, alterations in karyotype, morphology, and biochemical properties may occur. Recently, immortalized cell lines derived from primary cultures have been isolated in which the cells exhibit apparently stable phenotypes and genotypes and retain much of the differentiated function of the parent cell. However, it is difficult if not impossible to ascertain whether such immortalized cells may accurately model native tissue in all physiological aspects.
Dissociated primary cell culture is the most widely used in vitro system in neurobiology and much work has focused on obtaining cultures representative of neuronal populations in the brain, spinal cord, and dorsal root ganglion. The majority of studies on central and peripheral nervous tissue in primary culture have been performed using embryonic or neonatal tissues, as cells at early developmental stages possess greater potential for growth in culture (Crain, 1976). Both explant cultures and dissociated cell cultures from neonatal animals have been isolated to study specific cell types. For example, explants (Jessen et al., 1983) and cell cultures (Nishi and Willard, 1985) from the neonatal rat myenteric plexus have been developed to study the enteric nervous system (ENS). While primary cell cultures more faithfully reproduce the characteristics of native tissue than do immortalized cell lines, primary neuronal cell cultures derived from neonatal animals do not, in all cases, represent accurate models of adult neuronal systems. Specifically, ENS maturity is reached only at one month after birth and certain neuronal populations can only first be detected two to three weeks after birth (Matini et al., 1997). Further, nerve cell numbers per ganglia decrease by approximately half in the first two weeks of life (Schafer et al., 1999). Therefore, there is a need within the art of a supply of primary cells (v. primary cell cultures) from all developmental stages of an animal.
There are also other drawbacks associated with the use of primary cell cultures Primary cell cultures are inconvenient to work with in that they often require ordering and delivery of animals and/or timed pregnant animals and then difficult and expertise intensive dissection and dissociation procedures. Further, there are many scheduling difficulties that incur delays when carrying out primary cell culture work. This is particularly true in the case of primary neuronal cultures as they require fetal or neonatal substrate tissue. Also, the financial costs and resources required to maintain viable neuronal cultures at a research site can be high and often prohibitive. At present, there is currently no commercial source for mammalian neurons either as primary culture material or precursor culture material. This is due to the poor viability of neurons in culture and the inability to cryopreserve and transport neurons
Cryopreservation is employed widely for the prolonged storage of mammalian cell lines, tissue and primary non-neuronal cells such as hepatocytes (Swales et al 1996). However, cryopreservation of primary neurons has been limited to blocks of dissected CNS tissue which require thawing and dissociation of cells for culture (Hashimoto et al., 2000) A drawback of cryopreserving blocks of tissue is that the tissue must be thawed and cells of interest dissociated and isolated from the tissue and subsequently cultured. Processing of the cells requires significant expertise and the chances of injury or contamination of the neuronal cultures are high. U.S. Pat. No. 5,328,821 discloses cryopreservation solutions for tissue slices obtained from liver. Thus, there is a need for cryopreserved dissociated primary neuronal cells which may be easily thawed and cultured.
U.S. Pat. No. 6,140,123 discloses a method for preconditioning and cryopreservation of porcine hepatocytes cells harvested from a donor wherein the cells are suspended in a cryopreservation medium comprising DMSO, glutathione, adenosine, a calcium channel blocker and a cell nutrient matrix. The reference does not teach whether the compositions may be used for cryopreservation of primary dissociated neuronal cells. U.S. Pat. No. 6,140,116 discloses cell culture of porcine neural cells and methods for using the cells to treat neurological deficits due to neurodegeneration. The reference teaches that N-acetylcysteine and ascorbic acid may be used to counteract the adverse effects of oxidative stress during preparation for transplantation. However, there is no disclosure of the cryopreservation of neuronal cells. U.S. Pat. No. 5,972,923 discloses methods and compositions for enhancing the cytoprotective effects of polycyclic phenolic compounds through the synergistic interaction with anti-oxidants. The compositions disclosed may comprise a plurality of antioxidants including ascorbic acid, and glutathione. There is no disclosure as to whether any of the compositions may be employed to cryopreserve primary neuronal cells.
U.S. Pat. No. 5,849,585 discloses a method for enhancing the survival and proliferation of Schwann cells in cell culture using serum free culture medium comprising mitotic agents. In addition, other optional supplements may be added to the cell medium including vitamin E as an antioxidant and anti-transforming agent. U.S. Pat. No. 5,849,584 discloses a method for producing an expanded, primary, non-transformed cell culture wherein the cultured cells may be selected from glandular neuroblast, liver, adrenal cortex, oral mucosa, cartilage inner ear or bladder cells. The cells may be cultured in a medium comprising ascorbic acid between 30 and 125 mg/L. U.S. Pat. No. 4,927,762 discloses culturing cells in serum free media in combination with a non-toxic antioxidant (N-acetylcysteine, mercaptoproprionic acid, 2-mercaptoethanesulfonic acid, or thiolactate). WO 99/35242 teaches media compositions and culturing compositions for the growth and function of secretory cells. However, none of these documents disclose whether the compositions may be employed to cryopreserve primary dissociated neuronal cells.
The article entitled “Heme oxyenase immunoreactive neurons in the rat intestine and their relationship to nitrergic neurons” by Donat et al., Journal of the Autonomic Nervous System (1999) discloses isolation and culturing primary rat enteric neurons. However, the article does not teach cryopreservation of ENS or other cells from primary dissociated neuronal tissue.
There is a need in the art for cell culture and cell cryopreservation compositions which may be used to culture and cryopreserve primary neuronal cells. Further there is a need in the art for processes for the cryopreservation of primary neuronal cells. Further there is a need for cell cryopreservation compositions and methods of cryopreserving primary neuronal cell in high yield so that these cells may be shipped to distant destinations for use and study.
Further there is a need in the art to identify cryopreservation compositions and methods of culturing primary neuronal cells, such that cryopreserved cells are physiologically indistinguishable from noncryopreserved primary cells in regard to cell morphology, physiology and electrophysiology.
It is an object of the present invention to overcome disadvantages of the prior art.
The above object is met by a combination of the features of the main claims. The sub claims disclose further advantageous embodiments of the invention.